Reversal of Regioselectivity in Zinc-Dependent Medium-Chain Alcohol Dehydrogenase from Rhodococcus erythropolis toward Octanone Derivatives

Article abstract of DOI:10.1002/cbic.202000247

The zinc-dependent medium-chain alcohol dehydrogenase from Rhodococcus erythropolis (ReADH) is one of the most versatile biocatalysts for the stereoselective reduction of ketones to chiral alcohols. Despite its known broad substrate scope, ReADH only accepts carbonyl substrates with either a methyl or an ethyl group adjacent to the carbonyl moiety; this limits its use in the synthesis of the chiral alcohols that serve as a building blocks for pharmaceuticals. Protein engineering to expand the substrate scope of ReADH toward bulky substitutions next to carbonyl group (ethyl 2-oxo-4-phenylbutyrate) opens up new routes in the synthesis of ethyl-2-hydroxy-4-phenylbutanoate, an important intermediate for anti-hypertension drugs like enalaprilat and lisinopril. We have performed computer-aided engineering of ReADH toward ethyl 2-oxo-4-phenylbutyrate and octanone derivatives. W296, which is located in the small binding pocket of ReADH, sterically restricts the access of ethyl 2-oxo-4-phenylbutyrate, octan-3-one or octan-4-one toward the catalytic zinc ion and thereby limits ReADH activity. Computational analysis was used to identify position W296 and site-saturation mutagenesis (SSM) yielded an improved variant W296A with a 3.6-fold improved activity toward ethyl 2-oxo-4-phenylbutyrate when compared to WT ReADH (ReADH W296A: 17.10 U/mg and ReADH WT: 4.7 U/mg). In addition, the regioselectivity of ReADH W296A is shifted toward octanone substrates. ReADH W296A has a more than 16-fold increased activity toward octan-4-one (ReADH W296A: 0.97 U/mg and ReADH WT: 0.06 U/mg) and a more than 30-fold decreased activity toward octan-2-one (ReADH W296A: 0.23 U/mg and ReADH WT: 7.69 U/mg). Computational and experimental results revealed the role of position W296 in controlling the substrate scope and regiopreference of ReADH for a variety of carbonyl substrates.

Full text of DOI:10.1002/cbic.202000247

Combining Chemistry and Biology
Accepted Article
Title: Reversal of regioselectivity in zinc-dependent medium chain
alcohol dehydrogenase from Rhodococcus erythropolis toward
octanone derivatives
Authors: Mehdi D. Davari, Gaurao V. Dhoke, Yunus Ensari, Dinc Yasat
Hacibaloglu, Anna Gärtner, Anna Joëlle Ruff, and Marco
Bocola
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To be cited as: ChemBioChem 10.1002/cbic.202000247
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1/2020

ChemBioChem
10.1002/cbic.202000247
Reversal of regioselectivity in zinc-dependent medium chain
alcohol dehydrogenase from Rhodococcus erythropolis toward
octanone derivatives
Dr. Gaurao V. Dhoke§,[a] Dr. Yunus Ensari ,
§ [a,b]
Dinc Yasat Hacibaloglu,[a] Anna
[
a]
[a]
[a]
[a]
Gärtner, Dr. Anna Joëlle Ruff, Dr. Marco Bocola, Dr. Mehdi D. Davari*,
[a]Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074
Aachen, Germany
[b]Kafkas University, Faculty of Engineering and Architecture, Department of
Bioengineering, Kars, Turkey
§
G.V.D. and Y.E are joint first authors.
*Corresponding author:
Dr. Mehdi D. Davari
Lehrstuhl für Biotechnologie,
RWTH Aachen University
Worringerweg 3,
5
2074 Aachen (Germany)
Fax:(+ 49) 241-80-22387
E-mail: m.davari@biotec.rwth-aachen.de
Twitter: @mehdi_d_davari; @EnsariYunus; @DhokeGaurao; @RWTH.
1
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ChemBioChem
10.1002/cbic.202000247
Abstract
Zinc-dependent medium chain alcohol dehydrogenase from Rhodococcus
erythropolis (ReADH) is one of the most versatile biocatalyst for stereoselective
reduction of ketones to chiral alcohols. Despite the known broad substrate scope,
ReADH accepts only carbonyl substrates with either methyl or ethyl group adjacent to
carbonyl moiety which limits its use in synthesis of chiral alcohols which serve as a
building blocks for pharmaceuticals. Protein engineering to expand the substrate
scope of ReADH toward bulky substitutions next to carbonyl group (ethyl 2-oxo-4-
phenylbutyrate) opens up new routes in the synthesis of ethyl-2-hydroxy-4-
phenylbutanoate, an important intermediate for anti-hypertension drugs like enalaprilat
and lisinopril. We have performed computer-aided engineering of ReADH toward ethyl
2
-oxo-4-phenylbutyrate and octanone derivatives. W296 which is located in the small
binding pocket of ReADH restricts sterically the access of ethyl 2-oxo-4-
phenylbutyrate, 3- or 4-octanone toward the catalytic zinc ion and thereby limits
ReADH activity. Computational analysis was used to identify position W296 and Site
Saturation Mutagenesis (SSM) yielded an improved variant W296A with a 3.6 fold
improved activity toward ethyl 2-oxo-4-phenylbutyrate when compared to ReADH WT
(ReADH W296A: 17.10 U/mg and ReADH WT: 4.7 U/mg). In addition, the
regioselectivity of ReADH W296A is shifted toward octanone substrates. ReADH
W296A has a more than 16 fold increased activity toward 4-octanone (ReADH W296A:
0
.97 U/mg and ReADH WT: 0.06 U/mg) and a more than 30 fold decreased activity
toward 2-octanone (ReADH W296A: 0.23 U/mg and ReADH WT: 7.69 U/mg).
Computational and experimental results revealed the role of position W296 to control
the substrate scope and regiopreference of ReADH toward a variety of carbonyl
substrates.
Keywords: Alcohol dehydrogenase, Rhodococcus erythropolis, Protein engineering,
Homology Modeling, Molecular docking
Running title: Reversal of regioselectivity of ADH from Rhodococcus erythropolis
2
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ChemBioChem
10.1002/cbic.202000247
1
-Introduction
Secondary alcohol dehydrogenases (ADHs) are the enzymes belonging to the class
of oxidoreductases (E.C.1.1.1.1, also called as keto-reductases) and represent an
important class of biocatalysts because of their potential ability to stereospecifically
reduce prochiral carbonyl compounds. Stereoselectiveness is a unique characterstic
[
1]
among secondary alcohol dehydrogenases, which makes them of great interest for
the biocatalytic production of chiral alcohols as a building blocks for fine chemical
industries.[2] There are few described ADHs namely phenylacetaldehyde reductase
[
3]
from Cornynebacterium sp. Strain, Candida parapsilosis aldehyde dehydrogenase 5
cpADH5),[4] and ADH from Lactobacillus kefir,[5] which are cabable of reducing
ketones with bulky side chains. Recently, an ADH from the halophilic archaeon
(
[
6]
Haloferax volcanii (HvADH2) has been identified and engineered by rational design
approach to broaden its substrate scope towards the conversion of a variety of
aromatic substrates.[7] Furthermore, a bacterial β-ketoacyl-ACP reductase (FabG)
from Bacillus sp. ECU0013[8] and NADPH-dependent (S)-carbonyl reductase from
[
9]
Candida parapsilosis ATCC 7330 reduces ethyl 2-oxo-4-phenylbutanoate (EOPB) to
S)-ethyl 2-hydroxy-4-phenylbutanoate (S-EHPB). Optically pure EHPB is used in the
(
synthesis of an important intermediate for anti-hypertension drugs such as enalaprilat
and lisinopril[10]. In spite of having capability of reducing broad variety of substrate,
these enzymes differ in their substrate specificity and stereoselectivity. In addition,
they have insufficient operational long-term stability or limited substrate acceptance
which restricts their industrial applications. The ability to control the substrate
specificity and stereochemistry of ADH reactions is of increasing interest in
biocatalysis.[11] In recent years, a novel NADH-dependent ADH from Rhodococcus
erythropolis DSM 43297 (ReADH) has been emersed as a promising candidate for
use in industry.[5, 12] ReADH has a homodimeric structure and a molecular weight of
3
6,026 kDa per subunit.[12] It consists of 348 amino acids per subunit and belongs to
the zinc-dependent medium chain alcohol dehydrogenase (MDR) family. ReADH
follows the Prelog’s rule[13] which leads to formation of S-enantiomers with a high purity
(
e.e. ≥ 99 %).[12, 14] ReADH has a high stereo-, chemo- and regioselectivity towards
variety of carbonyl substrates including acetophenone derivatives, aliphatic ketones
and can withstand temperatures up to 65 C.[12, 14] Recently, Kasprzak et. al[15] has
reported the synthesis of 1-(S)-phenyl ethanol and ethyl (R)-4-chloro-3-
o
3
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ChemBioChem
10.1002/cbic.202000247
hydroxybutanoate employing a biphasic and a substrate-coupled cofactor
regeneration system. ReADH reduces carbonyl substrates that have either a methyl
or a ethyl group adjacent to carbonyl functionality[14] and a crystal structure has not yet
been reported.
In this work, we aimed to understand the structural determinants controlling the
substrate scope of ReADH. In order to expand the substrate scope of ReADH toward
bulkier substrates (ethyl 2-oxo-4-phenylbutyrate, 3- or 4-octanone), we reengineered
the smaller binding pocket of ReADH. Thereby we constructed a homology model of
ReADH and used for molecular docking of carbonyl substrates to identify positions
W296 as a key position in modulating the substrate access to the catalytic zinc center.
Identifcation was performed with our previously developed docking protocol.[4b]
2
2
. Methods
.1 Homology modeling
The amino acid sequence of ReADH (Accession no: C0ZXL4, 348 aa) was retrieved
from UniProt database[16] in FASTA format. Homology modeling of ReADH structure
was performed by using YASARA Structure Version 13.9.8[17] with the default settings
(
PSI-BLAST[18] iterations: 6, E value cutoff: 0.5, templates: 5, OligoState: 4). Template
structures was scored based on position specific scoring matrix (PSSM).[19] Two X-ray
templates were selected for hybrid modeling of ReADH structure (348 aa): Crystal
structure of alcohol dehydrogenase from Rhodococcus ruber (345 residues with
quality score 0.487, PDB ID: 3JV7,[20] resolution 2.0 Å) and crystal structure of the
alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix (343
residues with quality score 0.581, PDB ID: 1H2B,[21] resolution 1.62 Å). The developed
model is a hybrid homotetramer shown in Figure S1 in SI.
2
.2 Mechanism based substrate docking
The homodimeric structure of ReADH was used for substrate docking using
AutoDock4.2[22] within YASARA Structure Version 13.9.8.[17a] In this mechanism-
based docking protocol, reparameterization of catalytic zinc environment including
4
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ChemBioChem
10.1002/cbic.202000247
catalytic zinc ion and its coordinating residues (Cys38, His62, Asp153, and Water)
was carried out. Additionally, reaction mechanism based distance filter criteria
(distances D1, D2, and D3 shown in Figure S2 in SI) was applied. D1 signifies the
distance between carbonyl oxygen of the substrate to the catalytic zinc ion, D2
signifies the distance between the hydrogen (H) of NADH cofactor to the carbonyl
carbon (C) of substrate and D3 signifies the distance between carbonyl oxygen to the
(HO) group of Ser46. The detailed of substrate based docking protocol along with
applied distance constraint can be found elsewhere.[4b] For substrate molecular
docking protocol, bonded model of catalytic zinc ion with charge of +1.01 on catalytic
12
[23]
zinc ion and AM1-BCC charges for the substrate were used. Pymol was used to
prepare all the graphical images.
2
.3 Chemicals
All chemicals were purchased from Sigma (Steinheim, Germany), Aldrich (Steinheim,
Germany), Fluka (Steinheim, Germany), Merck (Darmstadt, Germany) and Roth
+
(
Karlsruhe, Germany) if not stated otherwise. NAD cofactor was purchased from
Sigma-Aldrich (Steinheim, Germany). Primers were ordered from Eurofins MWG
Ebersberg, Germany).
(
2
.4 Construction of Site-Saturation Libraries (SSM) of ReADH
The expression vector pKA1, harboring the (S)-alcohol dehydrogenase from
Rhodococcus erythropolis (ReADH),[12] was used as template for saturation
mutagenesis of position W296. Modified two step QuikChange Mutagenesis (QCM)
protocol was applied to generate mutant libraries. In the first step, two extension
reactions are performed in separate tubes; one containing the forward primer (5`-
ACAGTTCCGTATNNKGGTGCCCGC-3') and the other containing the reverse primer
(5'- GCGGGCACCMNNATACGGAACTGT -3'). After 3 rounds of extension, the two
reactions are mixed and amplification carried out for 15 cycles. Obtained PCR
products were digested with 20U DpnI (New England Biolabs, Frankfurt am Main,
Germany) in order to remove template DNA and purified with PCR purification kit
(
NucleoSpin® Gel and PCR Clean-up kit, Macharey-Nagel, Düren, Germany).
5
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ChemBioChem
10.1002/cbic.202000247
Libraries were transformed into E.coli BL21 Gold (DE3) and plated on LBCM agar
plates.
2
.5 Protein Expression in 96-Well Plates and Preparation of Crude Cell Extracts
Libraries were constructed by transferring single colonies from agar plate to 96-well
microtiter plates (Greiner Bio-One GmbH, Frickenhausen, Germany) filled with 150 µL
LB medium supplemented with 34 µg/ml chloramphenicol. Plates were tightly sealed
with insulation tape and incubated in a microtiter plate shaker (Multitron II, Infors
GmbH, Einsbach, Germany; 16 h, 37 °C, 900 rpm, 70 % humidity). For long-term
storage, 100 µl of 50 % (v/v) glycerol was added to each well and the libraries were
stored at -80 °C as master microtiter plate.
Expression in microtiter plates was performed by duplicating master plate with 96-pin
replicator to 150 µL of LB medium supplied with chloramphenicol (34 µg/ml) in 96-well
flat bottom microtiter plates (pre-culture). Plates were closed with lids, tightly sealed
with insulation tape and incubated for 16 h (37 °C, 900 rpm, 70 % relative humidity).
Then, 10 µL of the pre-culture were transferred to V-bottom microtiter plates
(
transparent polystyrene plate, Corning GmbH, Kaiserslautern, Germany) containing
50 µl terrific broth (TB) medium supplemented with chloramphenicol (34 µg/ml), trace
element solution (0,25 mL containing; 3.4 mM CaCl , 0.6 mM ZnSO , 0.6 mM MnSO
4.0 mM Na –EDTA, 61.8 mM FeCl , 0.6 mM CuSO and 0.8 mM CoCl in water), 0.1
mM IPTG, 0.1 g/L thiamine hydrochloride and 1 mM ZnCl . Plates were closed with
lids, tightly sealed and incubated in a microtiter plate shaker for 20 h (30 °C, 900 rpm,
1
2
4
4
,
5
2
3
4
2
2
7
3
0 % relative humidity). Expression cultures were harvested by centrifugation (4 °C,
220 g, 15 min) using an Eppendorf 5810R centrifuge (Eppendorf AG, Hamburg,
Germany) and stored at -20 °C until further use.
2
.6 96-well MTP activity assay for screening of SSM libraries.
The expressed variants in 96-well microtiter plates were taken out of the freezer and
kept at room temperature for 10 min. Cell lysates were prepared by resuspending each
cell pellet by pipeting in 150 µL lysis buffer (50 mM phosphate buffer pH 7.5, and 1 g/L
6
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ChemBioChem
10.1002/cbic.202000247
lysozyme). Cell lysis was performed by incubating suspended cells in a microtiter plate
shaker for 1 h (37 °C, 900 rpm, 70 % relative humidity) and followed by centrifugation
(4 °C, 3220 g, 20 min). Screening of SSM library was performed through monitoring
NADH depletion, result of ethyl 2-oxo-4-phenylbutyrate reduction to corresponding
alcohol, spectrophotometrically (Tecan sunrise, Männedorf, Switzerland). Reaction
setup contains 145 µl phosphate buffer pH 7.5, 50 µl of crude cell lysate, and 5 µl of
0
.5 M ethyl 2-oxo-4-phenylbutyrate in ethanol. The reaction was initiated by addition
of 50 µl of 1 mM NADH and absorption was monitored at 340 nm for 10 min (ɛ = 6200
1
/(M cm)).
2
.7 Flask Expression of ReADH Protein
For flask expression of ReADH enzyme, 4 ml of overnight culture grown in LB medium
with chloramphenicol (34 µg/ml; 37 °C, 250 rpm) was inoculated into 250 ml TB
medium supplemented with chloramphenicol (34 µg/ml) and trace element solution
(
0,25 ml containing; 3.4 mM CaCl
EDTA, 61.8 mM FeCl , 0.6 mM CuSO
7 °C, 250 rpm until OD600 reaches 0.6-0.8. Protein expression was induced by
addition of 0.1 mM IPTG and supplemented with thiamine hydrochloride (0.1 g/L) and
ZnCl (1 mM). Protein expression was carried out at 30 °C, 250 rpm for 20 – 22 h.
2
, 0.6 mM ZnSO
4
, 0.6 mM MnSO
4
, 54.0 mM Na –
2
3
4
and 0.8 mM CoCl
2
in water) and cultivated at
3
2
Cells were harvested by centrifugation (4 °C, 3220 g, 15 min) and stored at -20 °C
until further use.
2
.8 Activity determination of ReADH enzyme
ReADH enzyme was partially purified by heat treatment. For this procedure, 1 g cell
pellet was dissolved in 4 mL 50 mM phosphate buffer pH 7.5 and cell disruption was
performed by sonication for 5 min (30 sec. on + 30 sec. off, 40 % amplitude, SONICS,
Vibra cell, VCX-130 Frankfurt-Germany). Cell lysate containing soluble enzyme was
clarified by centrifugation (21300 g, 20 min, and 4°C). Cleared cell lysate was further
incubated at 65 °C for 15 min and centrifuged at 21300 g, 20 min, and 4 °C. 80 % of
the E. coli proteins could be removed by treating the cell crude extract for 15 min at
6
5 °C.[12] Obtained supernatant was used for activity measurements. ReADH activity
7
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ChemBioChem
10.1002/cbic.202000247
assays in cuvette-scale were performed in the Varian Cary 50 Bio UV/vis
spectrophotometer (Agilent Technologies, Darmstadt, Germany). 20 µL of substrates
in ethanol (10 mM final concentration) were mixed with 50 µL 5 mM NADH (0.25 mM
final concentration) and 830 µL 50 mM phosphate buffer at pH 8.0. The reaction was
started by addition of 100 μL of partially purified ReADH enzyme. NADH absorption at
3
40 nm was monitored for 10 min (ε = 6200 1/(M cm)). Protein concentration of the
heat treated lysate was determined using the Pierce™ BCA Protein Assay Kit
according to the manufactures instructions (Thermo Fisher Scientific, Darmstadt,
Germany). Heat-purified ReADH was diluted accordingly in order to obtain linear
absorbance change in the monitored time scale. The linear part of the measured
activity curve was used for calculation of enzymatic activity and stated in U/mg total
protein concentration in heat-purified lysate. All measurements were performed in
triplicate.
3
. Results and discussion
The main aim of the study was to understand the structural determinants for the
substrate scope of ReADH and its expansion toward bulky substrates for synthesis of
highly valuable chiral alcohols. First, we present the construction and evaluation of
ReADH homology model. Followed by molecular docking studies of ethyl 2-oxo-4-
phenylbutyrate, 2-, 3- or 4-octanone employing our previously developed mechanism
based docking protocol. In the next paragraph, in silico generation of the ReADH
W296A variant and results on docking studies are shown. Finally, the catalytic
performance of ReADH WT and ReADH W296A toward octanone derivatives and a
shift in regioselectivity of ReADH W296A are discussed.
3
.1 Structural modeling of ReADH
The main aim behind the construction of a homology model for ReADH is to get
insights into the substrate binding pocket of ReADH. Based on the sequence of
ReADH, hybrid homology model was constructed with two different templates (PDB
ID: 3JV7;[20] ADH from Rhodococcus ruber, and PDB ID: 1H2B;[21] ADH from
8
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ChemBioChem
10.1002/cbic.202000247
Aeropyrum pernix) using YASARA Structure Version 13.9.8.[17] Details of homology
modeling is discussed in SI.
On closer inspection of the active site of ReADH, it was observed that the catalytic
zinc ion is coordinated to C38, H62, and D153; the fourth coordination site is occupied
by acetic acid (adapted from template structure PDB ID: 3JV7). However, the
structural zinc ion is coordinated to four cysteine residues (C92, C95, C98, and C106).
The constructed homology model shown in Figure 1.
Figure 1. Homodimeric structure of ReADH modeled using YASARA Structure
Version 13.9.8.[17] with the docking pose of acetophenone. The catalytic active site
along with the zinc ion (orange sphere) and its coordinating residues (C38, H62, and
A153) along with NADH as a cofactor are shown. Substrate (acetophenone) is shown
in ball and stick representation. The residue S40 which is involved in proton relay
mechanism is also shown.
3
.2 Structure-guided analysis of the binding pocket of ReADH
The catalytic residues that are involved in the carbonyl reduction mechanism and
maintains the tetravalency of catalytic zinc ion were identified by comparing the
constructed ReADH homology model with the crystal structure of cpADH5 (PDB ID:
4
C4O).[25] The structural overlay of homology model of ReADH and cpADH5 is shown
in Figure S4 in SI. Root Mean Square Deviation (RMSD) was found to be 0.61 Å
indicating structural similarities between these two structures. It was found that the
binding pocket of cpADH5 is completely overlay to the constructed model of ReADH
9
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ChemBioChem
10.1002/cbic.202000247
and cpADH5 shares overall 30.70 % sequence identity (depicted in Figure S5 in SI).
The active site of the optimized model was further examined using YASARA Structure
Version 13.9.8.[17] Previous literatures have reported that the binding pocket of ADHs
belonging to zinc-dependent medium chain reductase family consists of small and
large binding pockets.[25b, 26] These binding pocket mainly determines substrate scope
and selectivity[27] of ADHs. The large binding pocket is formed because of the shape
of the entrance of substrate from the solvent media toward the catalytic zinc ion.
However, the small binding pocket is defined by W296 which acts as a gatekeeper for
smaller binding pocket. Conservation analysis showed that W296 is a 70 % conserved
residue in the family of zinc-dependent MDRs (depicted in Figure S6 in SI). The
surface view of the identified small and large binding pocket of ReADH is depicted in
Figure 2. The smaller binding pocket is constituted by the residues C38, D153, T157,
Y295, W296 and the larger binding pocket is constituted by the residues S40, F43,
I44, Y52, Y54, H62, and L119.
Figure 2. Binding pocket of ReADH consisting of small (red wireframe) and large
+
binding pocket (blue wireframe). NAD cofactor is shown as stick and catalytic zinc ion
as a orange sphere. Residues in the small (red color) and large (blue color) binding
1
0
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ChemBioChem
10.1002/cbic.202000247
pocket are shown as sticks. The coordinating residues (C38, H63, and D153) to
catalytic zinc ion can be seen in the figure.
The catalytic triad (Cys-His-Asp) which is coordinated to catalytic zinc ion is common
in both (ReADH and cpADH5). The carbonyl reduction mechanism in cpADH5 was
studied by Dhoke et. al[28] and firstly the hydride transfer takes place in carbonyl
reduction mechanism followed by the sequential events of proton transfers.
Considering the active site similarities in both the enzymes, we assumed a similar
carbonyl reduction mechanism for ReADH (depicted in Figure S7 in SI). The serine
residue (S46) which is involved in first proton transfer step (PT1) in cpADH5 is
positioned at the same place (S40) in ReADH. In addition to this, histidine residue
(H39) is also occupies the similar position and orients toward the riboxyl sugar moiety
of NADH in order to transfer third proton transfer (PT3) during the catalytic reduction
mechanism. A hydrogen bond network was observed in ReADH between the S40-
riboxyl sugar of NADH-H39 which is similar to the one in cpADH5.
3
.3 Molecular docking of selected substrates
Based on the substrate binding pockets in ReADH, we selected 2-octanone as a
candidate substrate for carrying out molecular docking studies. The rationale behind
selection of 2-octanone as a candidate substrate was that it has one small (methyl)
and one long (hexyl) alkyl chain along with carbonyl functionality. Along with this,
substrates which have shown poor activity against MDRs sharing structural similarities
in the active sites (cpADH5 and ReADH) were collected from literature[29] (listed in
Table 1). The goal is to understand the structure-function relationship using ReADH
as a model enzyme and furthermore to design ReADH improved variants. In the
literature, it was mentioned that the relative activity (% activity of acetophenone) of
these substrates (ethyl 2-oxo-4-phenylbutyrate (EOPB), 2-hyroxyacetopheone, and
3
-chloropropiophenone) are lower than 20 % for cpADH5.[29b] Similarly, (S)-carbonyl
reductase from Candida parapsilosis ATCC 7330 also shows low activity (1.17 ± 0.19
U/mg) towards EOPB.[9]
1
1
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ChemBioChem
10.1002/cbic.202000247
Table 1. Substrates used in this study along with their structures.
Substrate
Structure
Reason of
selection
Reference
ethyl 2-oxo-4-
Low activity for
cpADH5
Jakoblinnert
2013[29b]
phenylbutyrate (EOPB)
2
-hydroxyacetophenone
2HACP)
-chloropropiophenone
3CPP)
Low activity for
cpADH5
Jakoblinnert
2013[29b]
(
3
Low activity for
Jakoblinnert
2013[29b]
(
cpADH5
acetophenone (ACP)
Reference substrate Schubert 2001[30]
and Dhoke
2
015[4b]
propiophenone (PPP)
acetophenone
derivative
t. w.
t.w.
t.w.
2
3
-octanonone (2OCT)
-octanone (3OCT)
Based on ReADH
binding pocket
Studying ReADH
structure-function
relatioship
4
-octanone (4OCT)
Studying ReADH
structure-function
relationship
t.w.
t.w. this work
Additionally, acetophenone was included as a reference substrate as reported for
cpADH5.[4b, 30] Furthermore, propiophenone was included in order to determine the
accessable space in the small binding pocket. All the substrates used in this study are
listed in Table 1. The molecular docking was conducted using our previously
developed substrate based docking protocol.[26] The binding energy obtained from
molecular docking of these substrates is listed in Table S2 in SI. The docking pose of
2
OCT and 3OCT in the binding pocket of ReADH is depicted in Figure 3.
1
2
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ChemBioChem
10.1002/cbic.202000247
From the docking pose of 2OCT, it was revealed that the carbonyl oxygen group of
both substrates is at a distance of <2 Å (shown in Figure 3). This indicates stronger
binding of 2OCT to the catalytic zinc ion. As can be seen from the Figure 3, when the
good substrate 2OCT is in the binding pocket, it positions itself in such way that its
methyl group (next to carbonyl functionality) is settled in the small binding pocket.
Therefore, we assumed that substitutions at the W296 position could be beneficial for
substrates with varied alkyl chain lengths. Therefore, 3OCT (docking pose shown in
Figure 3) and 4OCT were additionally included as substrates. With ReADH WT, non-
catalytic binding modes of 4OCT and EOPB were obtained as 4OCT has butyl and
EOPB has ethyl acetate substitution next to their carbonyl functionality, respectively.
In both cases, the carbonyl oxygen was far away (D1˃4 Å) from the catalytic zinc ion.
Figure 3. Molecular docking pose of A) 2-octanone and B) 3-octanone in the binding
pocket of ReADH; substrates are shown in ball and sticks representations, whereas,
all the active site residues are shown in sticks representation. Catalytic zinc ion is
shown as orange sphere and residue W296 is shown as magenta sticks. Reciprocal
arrows show the distance (D1) between the catalytic zinc ion and carbonyl oxygen of
the octanone substrates.
Based on structured based protein engineering of ADH from Candida parapsilosis
(cpADH5; small binding pocket; residue W286), we showed that by modifying the
smaller binding pocket in MDRs, the substrate scope can be controlled.[31] This is
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ChemBioChem
10.1002/cbic.202000247
because the side chain of tryptophan residue (W296) forming the small binding pocket
introduces steric hindrance, which prevents high activity of ReADH toward bulky
substrates with larger substitution adjacent to their carbonyl functionality. A similar
study on ADH from Thermoanaerobacter brockii (TbSADH) has revealed that by
mutating the residues (I86A, and W110T) of binding pocket of ADHs not only changes
the shape of the binding pocket but also interfere with the substrate-enzyme
interactions.[27] This ultimately can help in increasing the substrate scope toward
bulkier substrates. Therefore, we designed in silico the ReADH W296A variant with an
assumption that by substituting W296 with a smaller amino acid, the catalytically
competent binding orientation of 4OCT and EOPB could be achieved. We increased
the space (1300 Å) in the binding pocket of ReADH W296A when compared to ReADH
WT (1050 Å). With the increase in space, we achieved the catalytic binding EOPB
and 4OCT as shown in Figure S8 in SI. Designed ReADH W296A variant was further
used for molecular docking of 4OCT and EOPB. From the docking experiment with
ReADH W296A variant, it was observed that for both the substrates (EOPB and
4
OCT), the catalytic zinc ion is in close proximity (distance D1<2 Å) to that of the
carbonyl oxygen indicating the possibility of activation of carbonyl oxygen by the
catalytic zinc ion. The docked pose of EOPB with ReADH WT and ReADH W296A
variant is shown in Figure 4. Afterwards, NADH consumption assay was used to
convert all these substrates.
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ChemBioChem
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Figure 4. Molecular docking pose of ethyl 2-oxo-4-phenylbutyrate (EOPB) in the
binding pocket of ReADH; (A) ReADH WT and (B) ReADH W296A variant; substrate
is shown in ball and sticks representations whereas all the active site residues are
shown in stick representation. Catalytic zinc ion is shown as orange sphere and
residue W296 and A296 are shown as magenta sticks. Reciprocal arrow shows
distance (D1) between catalytic zinc ion and carbonyl oxygen of the substrate.
3
.4 Screening of SSM W296 library toward ethyl 2-oxo-4-phenylbutyrate and
activity measurement of ReADH variants
Activity measurements of ReADH WT toward selected substrates EOPB, 2HACP,
3
CPP, ACP, PPP, 2OCT, 3OCT, and 4OCT were performed to gain molecular insights
and to evaluate the molecular docking simulations. ReADH WT was expressed and
partially purified through heat treatment; purity was checked by SDS-PAGE analysis
(Figure S9 in SI). Activity toward various substrates was determined for cleared cell
lysates, heat treated samples and no significant activity change was observed for both
samples (Figure S10 in SI). We could not determine activity accurately toward
2
HACP, 3CPP and PPP due to low solubility of substrates during activity
measurement, and thus they were not further considered.
As computational prediction shows that W296 is a key player for the catalytic binding
of EOPB, we generated a SSM library at position W296. The rationale behind
generation of SSM library at W296 position was to explore the full diversity and to gain
deeper knowledge and to understand structure-function relationship of ReADH.
One hundred eighty clones (≥ 95 % coverage[32] were screened through the NADH
depletion assay for reduction of EOPB. Ten beneficial clones were sequenced and
three different substitutions were obtained (alanine, glycine, and cysteine). Shake flask
expression was performed for all three variants and specific activities were determined
after partial purification. ReADH W296A variant showed a high activity toward EOPB
when compared to ReADH W296C and W296G. ReADH W296A, W296C, and
W296G variants showed 3.6, 3.0, and 1.9 fold increased specific activity when
compared to ReADH WT (ReADH WT: 4.7 U/mg; W296A:17.10 U/mg; W296C: 14.4;
and W296G: 9.1 U/mg). All three amino acids (alanine, glycine, and cysteine) have
side chains with a low steric demand when compared to the indole ring of tryptophan.
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ChemBioChem
10.1002/cbic.202000247
A similar effect was also observed for cpADH5 toward methyl 3-hydroxyhexanoate
and methyl 3-hydroxyoctanote when tryptophan residue at position 286 in small
binding pocket was substituted to alanine.[31] Kinetic results of ReADH W296A,
W296C, and W296G confirmed the computational analysis that Trp acts as a
gatekeeper to the small binding pocket. EOPB could not be accommodate in the
binding pocket of ReADH WT due to its size. Furthermore, specific activity toward
acetophenone which was used as reference substrate for cpADH5,[4b] was determined
for ReADH W296A, W296C, and W296G. Obtained variants showed a drastic activity
decreases toward acetophenone (ReADH WT 4.3 U/mg, W296A 0.1 U/mg activity).
Because W296A has shown to have the highest activity among the improved variants,
it was chosen for further analysis. Specific activities of the ReADH WT and W296A
were determined toward 2OCT, 3OCT, and 4OCT in order to elucidate the observed
binding of these substrates in the active site. ReADH WT shows a gradually decreased
activity toward 3OCT and 4OCT (See Figure 5). ReADH W296A variant has an
opposite preference; W296A’s activity gets increased by change of carbonyl oxygen
position on octanone chain. The ReADH W296A variant has more than 16 fold activity
increase toward 4OCT when compared to the ReADH WT. A shift in the
regioselectivity is observed for the ReADH W296A variant. Whereas ReADH WT
accommodates small chemical groups like a methyl group in acetophenones and 2-
octanone, the ReADH W296A variant accepts in its small binding pocket larger
chemical groups such as propyl moiety of 4OCT.
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Figure 5. Specific activities of ReADH WT and improved variants for reduction of
acetophenone, ethyl 2-oxo-4-phenylbutyrate, 2-octanone, 3-octanone, and 4-
octanone. All activity measurements were performed with heat-purified lysate at 5 mM
substrate concentration and NADH depletion was monitored at 340 nm.; WT, black;
W296A, red; W296C, blue and W296G, green. Specific activities of ReADH WT and
W296A variant for reduction of 2-octanone, 3-octanone, and 4-octanone are shown
on embedded figure. WT, black; W296A, gray.
3
.5 Structural determinants of ReADH regioselectivity
Different carbonyl substrates were selected and investigated experimentally to explore
full substrate scope of ReADH. As discussed earlier, ReADH has a small as well as
large binding pocket and smaller binding pocket restricts the entry of substrates which
has larger substitutuent than ethyl group next to the carbonyl functionality. There is a
significant similarity between binding pocket of cpADH5 and ReADH, therefore it is
highly likely that butyraldehydes[4b] can also be considered as the natural substrate of
ReADH, which has one carbon on smaller aliphatic chain. The selected substrate
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2
OCT orientates catalytically in the binding pocket of ReADH in such a way that its
smaller aliphatic chain having methyl group is positioned in the small binding pocket
as shown in Figure 3A. Therefore, it has been proposed that the substituting residue
W296 with smaller amino acid (e.g. alanine) increases the volume inside the small
binding pocket which gives information on the definition and the boundaries of this
pocket of ReADH WT toward the substrates with different chain lengths. Thus, 3OCT
and 4OCT from the same octanone class with different positioning of carbonyl
functionality were tested to understand the structure-function relationship of ReADH.
3
OCT and 4OCT along with 2OCT were tested experimentally and the measured
activity were depicted in Figure 5 and Figure S11 in SI.
ReADH WT shows higher activity toward 2OCT compared to ReADH W296A variant
as there is space only for methyl or ethyl group in the smaller binding pocket. In case
of 3OCT and 4OCT, ReADH WT showed less activity compared to 2OCT (depicted in
Figure 5) because of the increase in the side chain length. It was found that on
increasing the length of aliphatic side chain next to carbonyl moiety on octanone chain,
the activity of ReADH WT decreases. ReADH WT shows gradually decreased activity
toward 3OCT and 4OCT. Therefore, the smaller binding pocket of ReADH WT enzyme
was engineered to understand the substrate scope. The improved ReADH W296A
variant obtained after SSM at W296 position was used further for this purpose.
Molecular docking simulations also revealed that by substituting W296 position with
smaller amino acids, more space can be accessible for substrates having larger than
ethyl group next to the carbonyl functionality. This can be clearly seen from docking
pose of 4OCT depicted in Figure 6.
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ChemBioChem
10.1002/cbic.202000247
Figure 6. Molecular docking pose of 4-octanone in the binding pocket of ReADH;
ReADH W296A variant; substrate is shown in ball and sticks representation, whereas
all the active site residues are shown in sticks representation. Catalytic zinc ion is
shown as orange sphere and residue A296 are shown in magenta sticks. Reciprocal
arrow shows distance (D1) between catalytic zinc ion and carbonyl oxygen of the
substrate.
From our experimental data, it was found that the improved ReADH W296A variant
showed completely opposite trend in activity toward 2OCT, 3OCT, and 4OCT
compared to ReADH WT. The activity has increased by change of carbonyl oxygen
position. For 4OCT, the activity increases more than a 16 fold for ReADH W296A
variant compared to the ReADH WT. Inversion of the regioselectivity is observed for
the ReADH W296A variant; instead of favoring small chemical groups like a methyl
group in acetophenone and 2OCT, ReADH W296A variant favors larger chemical
groups (i.e. propyl) in its small binding pocket. We demonstrate that the introduced
substitutions in the smaller binding pocket induces systematic change in the
catalytically competent substrate binding and the interactions of the carbonyl oxygen
with the catalytic zinc ion and thus determining the stereopreferences and
regioselectivity of the ReADH variants. ReADH follows Prelog’s rule and convert
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ChemBioChem
10.1002/cbic.202000247
carbonyl compounds to their S-enantiomeric alcohols. [12, 14] However, generated
ReADH W296A variant possibly could show reversal in enantioselectivity for smaller
substrates accepted by ReADH WT, since ReADH W296A variant has larger substrate
binding pocket which allows the substrate to explore pro-(R) conformations with
catalytically active hydride transfer distances. This phenomenon was observed almost
in all previously engineered ketoreductases such as cpADH5 [31] and TbSADH [27, 33]
.
4
. Conclusions
Computational assisted ReADH engineering revealed that enlarging the smaller
binding pocket enables ReADH to convert bulkier substrates (ethyl 2-oxo-4-
phenylbutyrate , 3- or 4-octanone) with larger alkyl chains adjacent to their carbonyl
functionality. Molecular docking of substrates to ReADH WT showed that W296 is
critical in controlling the substrate scope. SSM study at W296 position yielded impoved
ReADH W296A variant which confirms our computational prediction. Substitution of
W296 with smaller amino acid provided sufficient space for the catalytically competent
binding of ethyl 2-oxo-4-phenylbutyrate and 4-octanone substrates.
From our point of view comprehensive study on ReADH provides a molecular basis to
expand substrate scope and regioselectivity of ReADH. This study would open novel
synthetic routes for synthesis of an important intermediate for anti-hypertension drugs
like enalaprilat and lisinopril. Based on the structural determinants, it is very likely that
the obtained reengineering knowledge can be transferred to other zinc-dependent
ADHs with small and large binding pocket.
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Abbreviations
ADH – Alcohol Dehydrogenase
ReADH – Alcohol Dehydrogenase from Rhodococcus erythropolis
cpADH5 – ADH5 from Candida parapsilosis
HT – Hydride transfer
PT - Proton transfer
EOPB - ethyl 2-oxo-4-phenylbutyrate
2
3
HACP - 2-hydroxyacetophenone)
CPP - 3-chloropropiophenone
ACP - acetophenone
PPP - propiophenone
2
3
4
OCT - 2-octanonone
OCT - 3-octanone
OCT - 4-octanone
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ASSOCIATED CONTENT
Supporting Information
Supporting information includes homotetrameric structure of ReADH, details of
structural modeling of ReADH, Catalytically applied distance filter criteria, table with
molecular docking energy of the substrates, sequence as well as structural alignment
of cpADH5 and ReADH, conservation analysis of ReADH followed by proposed
catalytic mechanism of ReADH, calculated binding pockets, SDS-PAGE analysis of
ReADH WT protein and Comparison of activity of crude cell lysate and partially purified
ReADH WT protein.
AUTHOR INFORMATION
Corresponding Author
m.davari@biotec.rwth-aachen.de (M. D. Davari)
Notes:
The authors declare no competing financial interest.
ACKNOWLEDGMENT
M.D.D and G.D. would like to acknowledge DFG SPP 1166 BioNoCo for financial
support. Y.E. thanks Kafkas University, Kars, Turkey for funding. Simulations were
performed with computing resources granted by JARA-HPC from RWTH Aachen
University under project RWTH0116.
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10.1002/cbic.202000247
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Graphical Abstract
A versatile ADH from Rhodococcus erythropolis (ReADH) accepts only carbonyl substrates with either
methyl or ethyl group adjacent to carbonyl moiety. Modified ReADH converts bulky substrates (ethyl 2-
oxo-4-phenylbutyrate) and opens up a new route for synthesis of ethyl-2-hydroxy-4-phenylbutanoate,
which is an important intermediate for anti-hypertension drugs like enalaprilat and lisinopril.
2
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